Identification of Differentially Expressed Genes by cDNA-Amplified Fragment Length Polymorphism in the Biocontrol Agent Pichia anomala (Strain Kh5).
ABSTRACT cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was used to identify genes potentially involved in biological control, by strain Kh5 (Pichia anomala), of Botrytis cinerea, an important post-harvest pathogen on apples. Strain Kh5 was grown in yeast nitrogen base (YNB) plus glucose (G medium) or YNB plus cell walls of B. cinerea (B medium). Thirty-five primer pairs were used in AFLP amplifications, resulting in a total of more than 2,450 bands derived from the mRNA of strain Kh5 grown in B medium. Eighty-six bands (3.5%) corresponded to genes upregulated in B medium compared with G medium. Of these 86 bands, 28 were selected, cloned, sequenced, and subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR) to confirm their differential expression. An appropriate housekeeping gene, G2, was selected and used to normalize the results of RT-PCR. Eleven genes presented an increased gene expression in the presence of B. cinerea cell walls (expression >1). Statistical analysis showed a significant increase for 5 of these 11 genes. The overexpressed genes show homologies to yeast genes with various functions, including beta-glucosidase, transmembrane transport, citrate synthase, and external amino acid sensing and transport. Some of these functions could be related to cell wall metabolism and potentially involved in mycoparasitic properties.
Massart
Sébastien
S
Plant Pathology Unit, FacultéUniversitaire des Sciences Agronomiques de Gembloux, Belgium.
Jijakli
M Haissam
MH